M. tuberculosis and M. bovis are important pathogens of man and animals. M. tuberculosis is thought to infect up to a third of the world's human population, remaining undetected during a latent phase of infection and reactivating to cause 10 million cases of tuberculosis and other diseases per year, resulting in 2 million deaths (Corbett et al. (2003) Arch. Intern. Med. vol. 163 pp 1009-1021). M. bovis, which has more than 99.9% sequence identity with M. tuberculosis, is the causative agent of bovine tuberculosis (BTB) and also causes disease in human. BTB represents a significant economic burden to the agricultural industries of various countries including the United Kingdom (Krebs (1997) “Bovine Tuberculosis in Cattle & Badgers” HMSO, London, United Kingdom).
The primary diagnostic test used in the control and surveillance of bovine TB is the tuberculin skin-test, a test that has remained in the forefront of TB diagnosis in both man and cattle for over 100 years. The development of the test arose following the preparation of the first ‘tuberculin’ by Robert Koch in 1890. Whilst Koch's tuberculin failed to live up to its initial claims of having curative properties, its diagnostic potential was quickly realised. The most common formats of the test used in cattle are the caudal fold test (CFT), the single intradermal cervical tuberculin test (SIT) and the single intradermal comparative cervical tuberculin (SICCT) test (Monaghan et al. (1994) Vet. Microbiol. vol. 40 pp 111-24). These test formats use a purified protein derivative (PPD) tuberculin prepared from a culture of M. bovis (PPD-B) as the primary diagnostic antigen. Additionally, the SICCT test includes the use of a M. avium derived PPD (PPD-A) to provide a measure of environmental sensitisation. It is the most specific of the tests (Plum (1931) Cornell Vet. vol. 21 pp 68-76; Stenius (1938) Veterinary Record vol. 50 pp 633-7) and is, therefore, the adopted test format in the UK.
In addition to skin tests, blood-based diagnostic assays that measure antigen-induced lymphokine production such as the interferon gamma (IFN-γ) test are also under consideration. The cytokine IFN-γ appears to be critical in the development of immunity to M. tuberculosis. For example, mice with a disrupted IFN-γ gene and also humans with a mutated IFN-γ receptor are highly susceptible to mycobacterial infections. However, specificity constraints are associated with the use of PPD in such assays. These arise due to the crude mixture of M. bovis proteins that PPD contains, many of which are cross-reactive with the BCG vaccine strain and environmental mycobacterial species such as M. avium and M. intracellulare. 
Bovine TB is a significant and ongoing problem in the UK. Cattle vaccination has been identified as one of the most promising long term UK control strategies (Krebs (1997) “Bovine Tuberculosis in Cattle & Badgers” HMSO) and the development of an efficacious vaccine continues to be a research priority. Currently, promising vaccines against bovine TB are based on heterologous prime-boost combinations that include the live attenuated M. bovis vaccine strain Bacille Calmette-Guerin (BCG) as one of their components (Hogarth et al. (2006) J. Pharm. Pharmacol. vol. 58 pp 749-57). However, as in humans, vaccination of cattle with BCG compromises the specificity of the tuberculin skin-test since PPD contains cross reactive antigens shared by both pathogenic and vaccine strains (Berggren (1981) Br. Vet. J. vol. 137 pp 88-94; Buddle et al. (1999) Clin. Diagn. Lab. Immunol. vol. 6 pp 1-5; Waddington & Ellwood (1972) Br. Vet. J. vol. 128 pp 541-52). Therefore, the development of diagnostic tests that can differentiate vaccinated from infected animals, so-called DIVA tests, are an essential pre-requisite to allow the inclusion of BCG-based vaccination as part of bovine TB control strategies.
Previous studies have demonstrated that diagnostic reagents which distinguish between vaccinated and infected cattle can be developed using specific, defined antigens that are present in virulent M. bovis but absent from the BCG. Genetic analysis of BCG has revealed that several large genomic regions have been deleted during attenuation and subsequent prolonged propagation in culture. These regions have been characterised and antigens from one of these regions, RD1, have been studied extensively in several species including humans and cattle. For example, it has been demonstrated that protein or polypeptide cocktails composed of two RD1 region antigens, ESAT-6 and CFP-10, can be used to distinguish between M. bovis infected and BCG-vaccinated cattle.
The practical application of such DIVA reagents have so far been largely realised through their use in blood-based interferon-.gamma. (IFN-.gamma.) release assays (IGRAs). For example, WO2009/060184 disclosed several polypeptides including epitopes from Rv3615c which were found to be useful to detect M. bovis infection, using such an assay. This polypeptide has also been confirmed as useful to detect M. tuberculosis infection in humans (Millington et al. (2011) Proc. Natl. Acad. Sci. USA vol. 108 pp 5730-5735). Rv3615c is referred to herein using the M. tuberculosis genome annotation. In the M. bovis genome, it is annotated as Mb3645c.
Given the high level of familiarity and wide-spread application of the tuberculin skin-test by veterinarians and clinicians, a DIVA skin-test format would provide a valuable additional test platform. This might especially be the case where the logistics of access to laboratory-based resources is problematic. It is also notable that, in recent years, there has also been renewed interest in a skin-test based DIVA test for human TB with several reports demonstrating the skin-test potential of ESAT-6 (Aggerbeck & Madsen (2006) Tuberculosis (Edinb.) vol. 86 pp 363-73; Arend et al. (2000) J. Infect. Dis. vol. 181 pp 1850-4; Wu et al. (2008) Clin. Exp. Immunol. vol. 152 pp 81-7). However, Whelan et al. (Infect. Immun. (2003) col. 71 pp 6420-6425) found that ESAT-6 was not effective on its own as a skin test reagent in cattle assessed using measurement of skin induration at the injection site. The addition of a synthetic bacterial lipopeptide adjuvant was required before ESAT-6 proved useful in cattle. It is preferable to avoid the use of adjuvants in a skin test reagent, since repeat skin test injections (as is required to monitor the health of, for example, a herd of dairy cattle) may lead to the sensitisation of non-tuberculosis infected animals, so that the skin test would cease to be useful to differentiate between infected animals and uninfected but vaccinated animals.
The present invention accordingly addresses the problem of providing discriminatory diagnostic reagents for the detection of mycobacterial infections using a DIVA skin-test format.